title: Transition-state stabilization in Escherichia coli ribonuclease P RNA-mediated cleavage of model substrates.
abstract: We have used model substrates carrying modified nucleotides at the site immediately 5' of the canonical RNase P cleavage site, the -1 position, to study Escherichia coli RNase P RNA-mediated cleavage. We show that the nucleobase at -1 is not essential but its presence and identity contribute to efficiency, fidelity of cleavage and stabilization of the transition state. When U or C is present at -1, the carbonyl oxygen at C2 on the nucleobase contributes to transition-state stabilization, and thus acts as a positive determinant. For substrates with purines at -1, an exocyclic amine at C2 on the nucleobase promotes cleavage at an alternative site and it has a negative impact on cleavage at the canonical site. We also provide new insights into the interaction between E. coli RNase P RNA and the -1 residue in the substrate. Our findings will be discussed using a model where bacterial RNase P cleavage proceeds through a conformational-assisted mechanism that positions the metal(II)-activated H2O for an in-line attack on the phosphorous atom that leads to breakage of the phosphodiester bond.
authors.affiliation: Department of Cell and Molecular Biology, Box 596, Uppsala University, SE-751 24 Uppsala, Sweden, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA and Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, SE-751 24 Uppsala, Sweden.
authors.forename: Shiying
authors.initials: S
authors.lastname: Wu
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.forename: Guanzhong
authors.initials: G
authors.lastname: Mao
authors.forename: Stefan
authors.initials: S
authors.lastname: Trobro
authors.forename: Marek
authors.initials: M
authors.lastname: Kwiatkowski
authors.forename: Leif A
authors.initials: LA
authors.lastname: Kirsebom
mesh_terms: D001483:Base Sequence; D055162:Biocatalysis; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry; D021122:Protein Subunits / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D059367:RNA Cleavage; D043262:Ribonuclease P / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D021122:Protein Subunits; D012313:RNA; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D010100:Oxygen